Peptides having an amino acid sequence from the fimbrial protein of porphyromonas gingivalis and their uses

ABSTRACT

There are provided peptides (each having 5 to 10 amino acid residues) corresponding to fragments derived from the amino acid sequence of the 41 kD sub-unit protein constituting the fimbriae of Porphyromonas gingivalis, or salts of the peptides. Further provided are compositions for diagnosis of periodontal disease containing them, vaccines for prophylaxis of periodontal disease containing part of them, and the oral compositions for prophylaxis or treatment of periodontal disease containing antibodies obtained by immunizing animals with the peptides. 
     Peptides are provided which are lowered in nonspecific reactivity and can be used more safely for the above compositions, compared to the above protein and peptides therefrom having 20 amino acid residues.

This application is a 371 of PCT/JP94/01589 filed Sep. 28, 1994.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides corresponding to fragmentsderived from the amino acid sequence of the 41 kD subunit proteinconstituting the fimbriae of Porphyomonas gingivalis, and their uses.More specifically, the invention relates to peptides acting as antigenswhich antigen-antibody react with antibodies against the 41 kD subunitprotein, and uses of the peptides for detection of specific antibodies,etc. in the serum, saliva and gingival crevice fluid of patients withperiodontal disease, and for prophylactic agents and treating agents ofperiodontal disease.

2. Description of Related Art

Periodontal diseases are classified into gingivitis and periodontitis,and further, periodontitis includes adult periodontitis, localizedjuvenile periodontitis, etc., but actually, 90% or more of periodontitisis occupied by adult periodontitis. These periodontal diseases arediseases including inflammations of gingiva, bleeding, drainage,formation of periodontal pockets, destruction of periodontal membranes,absorption of alveolar bones, and lability or loss of teeth. Variousbacteria exist at the lesions of these periodontal diseases, and amongthem, Porphyomonas gingivalis is considered to be a mainperiodontopathic organism, and remarkable increase of the bacterium isobserved at the lesions of periodontal diseases particularly adultperiodontitis.

At present, methods for treating periodontal disease are not perfectlyestablished, and it is considered to be the most important to findperiodontal disease as early as possible, grasp its pathologic statesaccurately, and make appropriate treatments, and further, prophylaxis ofperiodontal disease by development of vaccines for periodontal diseaseis desired.

First, as to diagnosis of periodontal disease, a diagnostic method forfinding periodontal disease as early as possible and grasping itspathologic state accurately has been expected, but actually, reliablediagnostic drugs for periodontal disease has not yet been developed.However, if ventured to be mentioned, as a means for diagnosingperiodontal disease through paying attention to periodontopathicorganisms, there are various methods for knowing the presence or numberof these periodontopathic organisms. Further, there have been proposedmethods to assay a specific antibody against a periodontopathic organismand utilize the result for diagnosis of periodontal disease. Stillfurther, methods have been proposed comprising assaying an inflammatoryproduct in the gingival crevice fluid of a patient with periodontaldisease.

There are various methods for knowing the presence or number of theseperiodontopathic organisms. For example, it was conducted to cultureperiodontopathic organisms in the dental plaques, gingival crevicefluid, saliva, etc. of a patient with periodontal disease in a bloodagar medium under an anaerobic condition, and investigate detailedbiochemical properties of the resultant various colonies, and therebydetect the periodontopathic organisms (Loesche, W. J., Syed, S. A.,Schmidt, E. and Morrison, E. C.: J. Periodont. 56, 447-456, 1985, etc.).

There has been conducted Gram staining, under microscopic observation,of periodontopathic organisms in dental plaques, gingival crevice fluid,saliva, etc. of a patient with periodontal diseases, or there has beenmade an examination under a dark-field microscope (Listgarten, M. A. andLevin, S.: J. Clin. Periodontol. 8, 122-138, 1981, etc.). There has alsobeen carried out the detection of a periodonto-pathic organism bycombining an antibody against it with a fluorescent dye (Zambon, J. J.,Bochacki, V. and Genco, R. J.: Oral Microbiol. Immunol. 1, 39-44, 1986).

Further, it has been conducted to detect periodontopathic organisms inthe dental plaques, gingival crevice fluid, saliva, etc. of a patientwith a periodontal disease, according to an immunological method such asenzyme-linked immunosorbent assay (ELISA method), using antibodiesagainst the respective periodontopathic organisms (see, Zambon, J. J.,Bochacki, V. and Genco, R. J.: Oral Microbiol. Immunol. 1, 39-44, 1986;Japanese Laid-open Patent Publication No. 159762/1988; ibid.107970/1990; ibid. 212061/1992; ibid. 355339/1992; ibid. 10954/1993; andEP 239,776 A).

Further, it has been conducted to pay attention to enzymes produced byperiodontopathic organisms, and detect the periodontopathic organisms inthe dental plaques, gingival crevice fluid, saliva, etc. of a patientwith periodontal disease, using their enzymatic activities as an index(see Loesche, W. J., Oral Microbiol. Immunol. 1, 65-70, 1986; JapaneseLaid-open Patent Publication No. 144997/1989; ibid. 144998/1989; ibid.499/1990; and ibid. 229198/1992).

Recently, it has also been conducted to pay attention to genes ofperiodontopathic organisms, and detect the periodontopathic organisms inthe dental plaques, gingival crevice fluid, saliva, etc. of a patientwith periodontal disease, using a DNA probe method (DMDx (trademark),Biotechnica Diagnostics Co.; and see Japanese Laid-open PatentPublication No. 135096/1990; and ibid. 502640/1991). Further, it isbecoming possible to detect an extremely slight amount of cells,according to the PCR (polymerase chain reaction) method.

However, diagnostic methods, as above-mentioned, for periodontal diseaseto know the presence and number of periodontopathic organisms are usableonly for diagnosis of pathogenesis, and are insufficient for graspingthe pathologic state of a patient. As a diagnostic method which makes apair therewith, and is for knowing the response of a patient as a hostto periodontopathic organisms and thereby grasping the pathologic stateof the patient, there can be thought a diagnostic method for periodontaldisease to know a specific antibody against a periodontopathic organism,and further its class and subclass. A diagnostic method for periodontaldisease to know the presence and number of periodontopathic organisms isalso, of course, important, and it is said that it is important toconduct appropriate diagnosis of periodontal disease by combining bothmethods.

As one of such diagnostic methods, a method is proposed to assay thespecific antibody titer against a periodontopathic organism of a patientwith adult periodontitis by the ELISA method, and diagnose theperiodontal disease of the patient from both of the results and theresults of dental checkups (see Mouton, C., et al.: Infection andImmunity 31, 182-192, 1981; Japanese Laid-open Patent Publication No.162753/1986; and ibid. 107969/1990).

Further, recently, a possibility that a diagnostic method forperiodontal disease is effective based on not only increase of aspecific antibody against a periodontopathic organism of a patient withperiodontal disease, but the fact that the subclass of the specificantibody changes from IgG₁ to IgG₄ together with change of thepathologic state of the periodontal disease is proved by the ELISPOTmethod to detect antibody-producing cells against periodontopathicorganisms in the gingiva tissue of a patient of adult periodontitis (T.Ogawa et al.: Clin. exp. Immunol. 76, 103-110, 1989).

When increase of a specific antibody against a periodontopathic organismof a patient with periodontal disease, and further change of its classor subclass are checked, an antigen used for detection becomesnecessary. In the above usual methods, entire cells of aperiodontopathic organism, or cell surface components such aslipopolysaccharides and fimbriae as an antigen have been used asantigens. However, it is difficult to obtain a large amount of pureantigen by the procedure of separation and purification, and manynon-relevant reactions take place thereon, and thus, they are notdesirable for accurately checking increase of specific antibodiesagainst the periodontopathic organisms of a patient with periodontaldisease, and further their classes or subclasses.

BBRC. 180, No. 3, 1335-1341 (1991) reports the immunogenicity of asynthetic peptide composed of 20 amino acid residues in the 41 kDsubunit protein molecule constituting the fimbriae of Porphyomonasgingivalis.

Further, it is disclosed in Japanese Laid-open Patent Publication No.135096/1990 to use a component derived from a periodontopathic organismin a patient with periodontal disease for detecting a specific antibodyagainst the periodontopathic organism, but the antigenic protein derivedfrom Porphyomonas gingivalis disclosed therein utterly differs from thefimbria of Porphyomonas gingivalis as a substance, and further, is notdesirable because it is troublesome to prepare an antigen as an entireprotein. As one of other diagnostic methods for periodontal disease,there is a method to assay an inflammatory product. For example, amethod is proposed to assay collagenase (Periocheck (trademark):Advanced Clinical Technologies, Westwood, Mass.), a cathepsin-likeactivity (Progno Stick: Dentsply Corp., York Pa.), or glucuronidase(Abbott Laboratories, North Chicago, Ill.). However, these methods haveproblems in specificity.

In Japanese Patent Publication No. 13205/1991, Japanese laid-open PatentPublication No. 140527/1986 and Japanese laid-open Patent PublicationNo. 132428/1993, it is proposed to use the fimbriae of Porphyomonasgingivalis as a prophylactic vaccine against periodontitis (orperiodontal disease). However, there are many problems in using fimbriaeseparated and purified according to methods mentioned therein. Namely,when the fimbriae are used after separation and purification, it isthought that substances other than the fimbriae, for example, cellularcomponents other than the fimbriae such as lipopolysaccharides which arepyrogens mingle therein, and the fimbriae are not desirable as vaccines.As understood from that it is shown in BBRC. 180, No. 3, 1335-1341(1991) that these fimbriae themselves exhibit various biologicalactivities, there is a possibility that use of whole of these fimbriaecauses actions other than the action as a vaccine, for example, aprophlogistic action and a pyrogenic action, which is undesirable.

Incidentally, the antigenic protein derived from Porphyomonasgingivalis, which is exhibited to be used as a vaccine in JapanesePatent Publication No. 135096/1990, differs from the peptide of thefimbriae of Porphyomonas gingivalis in the invention in amino acidsequence, and it is not desirable to use the entire antigenic proteinbecause preparation of the vaccine is troublesome and there is apossibility that it causes actions other than the action as a vaccine,for example, a prophlogistic action and a pyrogenic action. Further, thesynthetic peptide of the fimbriae of Porphyomonas gingivalis shown inAdv. Exp. Med. Biol. 327, 255-262, 1992 differs from the peptide of thefimbriae of Porphyomonas gingivalis in the invention in region, and itis not desirable, as in the above case, to use the entire proteinbecause preparation of the vaccine is troublesome and there is apossibility that it causes actions other than the action as a vaccine,for example, a prophlogistic action and a pyrogenic action.

An oral cavity composition containing an antibody obtained byimmunization with the whole cell or fimbrial protein of Porphyomonasgingivalis is proposed in Japanese laid-open Patent Publication No.142915/1985, ibid. 277632/1986, ibid. 289024/1986, ibid. 417/1987, ibid.313438/1989, ibid. 53458/1990, ibid. 53716/1990, ibid. 218620/1990,ibid. 59736/1992, ibid. 59737/1992 and Japanese Patent Publication No.63865/1992. However, antibodies against the whole cell or fimbrialprotein prepared by these methods possibly cause cross reaction withsubstances other than the desired antigen, and are not desirable for thepresent objects.

Thus, the object of the invention lies in providing peptides capable ofeffective detection of increase of specific antibodies againstperiodontopathic organisms in the patient with periodontal disease, andfurther change of their classes or subclasses, and a composition such asa peptide for diagnosis of periodontal disease. Another object of theinvention lies in providing a vaccine for prophylaxis of periodontaldisease containing a peptide having substantially no side-effect otherthan the desired vaccine action, among these peptides, and an oralcavity composition for prophyl axis or treatment of periodontal diseasecontaining an antibody obtained by immunizing an animal with a peptidethus selected.

SUMMARY OF THE INVENTION

For solving the above problems, the present inventors have maderesearches into, on various synthetic peptides corresponding tofragments derived from the amino acid sequence of the 41 kD subunitprotein constituting the fimbriae of Porphyomonas gingivalis,antigen-antibody reactivity between the synthetic peptides andantibodies against the above protein and biological activities of thesynthetic peptides. As a result, they found that peptides correspondingto the amino acid sequence and composed of consecutive 5 to 10 aminoacid residues unexpectedly have very excellent characteristics. Namely,although, as mentioned above, synthetic peptides corresponding tofragments derived from the amino acid sequence of the above protein andeach having 20 amino acid residues are disclosed in BBRC. 130, No. 3,1335-1341, 1991, the present inventors ascertained that the peptides ofthe invention are lowered in nonspecific reactivity and therefore, canbe used much more safely in various use fields, compared to the abovesynthetic peptides, and completed the invention.

Thus, according to the invention is provided a peptide corresponding toa fragment derived from the amino acid sequence of the 41 kD subunitprotein constituting the fimbriae of Porphyomonas gingivalis, thepeptide being characterized in that the fragment is selected fromfragments each composed of 5 to 10 consecutive amino acid residues, or aderivative thereof or a salt of the peptide or derivative.

According to a preferred embodiment of the invention, there is providedthe above peptide wherein the above fragment is selected from fragmentseach composed of consecutive at least 5 amino acid residues of thefollowing amino acid sequences (see SEQ ID NOs: 1 to 9 in Sequencelisting) or a derivative thereof or a salt of the peptide or derivative.

    Glu Asn Ala Thr Lys Val Glu Asp Ile Lys                                                                 (SEQ ID                                                 NO: 1);                                                                      - Glu Val Lys Ala Leu Thr Thr Glu Leu Thr (SEQ ID                             NO: 2);                                                                       - Ala Glu Asn Gln Glu Ala Ala Gly Leu Ile (SEQ ID                             NO: 3);                                                                       - Ala Ala Gly Leu Ile Met Thr Ala Glu Pro (SEQ ID                             NO: 4);                                                                       - Thr Gly Ser Leu Thr Thr Phe Asn Gly Ala (SEQ ID                             NO: 5);                                                                       - Thr Phe Asn Gly Ala Tyr Thr Pro Ala Asn (SEQ ID                             NO: 6);                                                                       - Gly Phe Tyr Val Leu Glu Asn Asp Tyr Ser (SEQ ID                             NO: 7);                                                                       - Ala Asn Gly Gly Thr Ile His Pro Thr Ile (SEQ ID                             NO: 8);                                                                       - and                                                                         - Glu Gly Lys Thr Tyr Tyr Pro Val Leu Val (SEQ ID                             NO: 9).                                                                

According to another embodiment of the invention, there is provided acomposition for diagnosis of periodontal disease containing one or acombination of two or more of the peptides or derivatives thereof orsalts of the peptides or derivatives.

According to still another embodiment of the invention, there areprovided a vaccine for prophylaxis of periodontal disease comprising oneor a combination, as its main ingredient, of two or more selected fromthe peptides or derivatives thereof or salts of the peptides orderivatives, the one or the two or more being characterized in that thepeptide is selected from fragments each composed of consecutive at least5 amino acid residues in the amino acid sequence represented by SEQ IDNO: 1 or 7 in Sequence listing, and pharmaceutically acceptablecarriers, and use of the above fragment for preparing such a vaccine,and a treatment method for periodontal disease which comprises a step toadminister the vaccine to a mammal.

According to still another embodiment of the invention, there isprovided an oral cavity composition for prophylaxis or treatment ofperiodontal disease containing an antibody obtained by immunizing ananimal with one selected from the above peptides or derivatives thereofor salts of the peptides or derivatives, the one being characterized inthat the peptide is selected from fragments each composed of consecutiveat least 5 amino acid residues in the amino acid sequence represented bySEQ ID NO: 1 or 7 in Sequence listing.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT(S)

The amino acid sequence of the 41 kD subunit protein constituting thefimbriae of Porphyomonas gingivalis, mentioned in the invention, meansthe sequence published in D. P. Dickinson et al., J. Bacteriol. 170, No.4, 1658-1665, 1988.

Thus, the peptides of the invention include all peptides so long as theyare peptides corresponding to fragments derived from the above aminoacid sequence, composed of consecutive 5 to 10 amino acid residues, andfit for the objects of the invention. Among these peptides, those havinga region exhibiting high antigenicity when reacted with serum of apatient with periodontal disease are particularly preferred in view ofthe objects of the invention, and as specific examples thereof, therecan be mentioned peptides of various chain lengths having consecutive atleast 5 amino acid residues in the amino acid sequences of the above SEQID NOs: 1 to 9.

These peptides of various chain lengths can be used as an antigen in theinvention because of having 5 amino acid residues at the smallestnumber, and the above nonspecific reactivity can be lowered by limitingthe peptides up to those having 10 amino acid residues at the largestnumber. Therefore, as specific examples of the peptides of theinvention, there can be mentioned peptides composed of consecutive 5, 6,7, 8, 9 or 10 amino acid residues, in the amino acid sequences of SEQ IDNOs: 1 to 9. For example, as an example of preferred specific peptidesamong those having 5 amino acid residues, there can be mentioned onerepresented by the following SEQ ID NO: 10 in Sequence listing:

    Glu Asn Ala Thr Lys                                        (SEQ ID NO: 10).

The derivative in the invention means a substance obtained by binding tothe peptide protective group(s), functional group(s), a spacer, acarrier or the like. More specifically, as examples of derivativeshaving a protective group, there can be mentioned a derivative whereinthe N-terminus of a peptide of the invention is protected with an acetylgroup, an urethane group or the like, and a derivative wherein theC-terminus of a peptide of the invention is protected with an amidogroup, an ester group or the like. Further, when the peptides arechemically bound, directly or indirectly, to solid phases or carriers,the amino group, carboxyl group or the like of the peptides themselvescan be utilized, but for utilizing a phenol group or a sulfhydryl group,a substance obtained by introducing tyrosine or cysteine into a peptideof the invention can also be used, and is also included in thederivative of the invention. Further, when an antibody binds to apeptide of the invention, a spacer can be introduced for the purpose ofmaking the antibody not easily suffer steric hindrance, and a derivativewherein such a spacer is introduced is also included in the derivative.As specific examples of such spacers, there can be mentionedα,ω-diaminoalkanes (n=2-12); α-amino-ω-carboxyalkanes (n=2-10);3,3'-diaminodipropylamine; succinyl-3,3'-diaminopropylamine;m-aminophenols to which bisdiazobenzidine is bound; Gly-Gly-Gly;Gly-Gly-Tyr; succinyl-1,3-diaminopropan-2-ol; polylysine, etc.

As the derivatives of the invention wherein a carrier is bound, therecan be mentioned derivatives wherein lysine used also as the spacer, oneof various polymers, bovine serum albumin or the like is bound to apeptide of the invention; derivatives wherein bovine serum albumin,tetanus toxoid, ovalbumin, keyhole limplet hemocyanin, thyroxin bindingglobulin, γ-globulin, a polysaccharide or the like, which is used when apeptide of the invention is used as an immunogen, is bound to thepeptide.

As salts of the peptides or derivatives, when the peptides themselves ortheir derivatives have a primary, secondary or tertiary amino group,there can be mentioned acid addition salts, for example, salts with aninorganic acid capable of giving a salt therewith such as hydrochloricacid, sulfuric acid, phophoric acid or pyrophophoric acid, salts with anorganic acid capable of giving a salt therewith such as acetic acid,lactic acid, palmitic acid, stearic acid, propionic acid, citric acid,tartaric acid, malic acid, ascorbic acid, oxalic acid, methanesulfonicacid, benzenesulfonic acid or p-toluenesulfonic acid. Further, when thepeptides themselves or their derivatives have a carboxyl group or asulfonyl group, there can be mentioned salts, for example, alkali metalsalts thereof such as sodium salts and potassium salts, alkaline earthmetal salts thereof such as calcium salts and magnesium salts, ammoniumsalts, triethylamine salts, etc.

Although it is possible to obtain the peptides of the invention bylimited decomposition of the 41 kD subunit protein constituting thefimbriae of Porphyomonas gingivalis, it is advantageous to obtain themby a peptide synthesis method known per se. As such peptide synthesismethods, there can, for example, be mentioned chemical synthesis methods[solid phase synthesis method and liquid phase synthesis method (seeJikken Kagaku Koza (Courses of Experimental chemistry) 22, Yuki Gosei(Organic synthesis) IV, pp 258-309, etc., Published on Nov. 30, 1992 byMaruzen Co., Ltd.)] and methods according to genetic engineering.

In this occasion, the synthesis can be conducted using MULTI-PIN PEPTIDESYNTHESIS KIT (CHIRON MIMOTOPES PTY LTD.: Australia) and a Model 9050Peptide synthsizer (Millipore corporation).

Derivatives from peptides thus obtained can be obtained by chemicalmodification means usually used in the technical fields of peptidechemistry. For example, when an aforesaid carrier is chemically bound tosuch a peptide, a method can be utilized to use glutaraldehtde, a watersoluble carbodiimide, succinimide or the like.

For detecting the specific antibody of a patient with periodontaldisease, the above peptides or derivatives thereof or salts of thepeptides or derivatives can be used alone or in a combination of two ormore. As immunological means usable for the detection, known methods canbe used, and as examples thereof, there can be mentioned an ELISAmethod, an RIA method, fluorescent antibody technique, chemicalluminescence antibody technique or the like wherein a polystyrene plate,a glass filter, magnetic particles, a latex or the like is used as thesolid phase, and the specific antibody of a patient of periodontaldisease is detected using a labeled secondary antibody; animmunoagglutination method wherein sheep erythrocytes, a latex or thelike is used as the solid phase; immunological nephelometry wherein alatex or the like is used as the solid phase; Western blot techniquewherein a nitrocellulose membrane or the like is used as the solidphase; etc. Also as to methods to immobilize a peptide, derivativethereof or salt of the peptide or derivative, as mentioned above, on asolid phase, various known methods can be utilized, and there can, forexample, be mentioned a method to physically adsorb it, a method tochemically immobilize it using glutaraldehyde or the like.

Further, immobilization of the antibody of a patient with periodontaldisease on a solid phase can be conducted using an anti-human antibodyor by physical adsorption, and after the immobilization, variousimmunological methods using a labeled peptide can be utilized.

When a peptide of the invention is used as a vaccine for prophylaxis ofperiodontal disease, it can also be adsorbed on a carrier protein suchas, bovine serum albumin, tetanus toxoid or the like. In addition, thepeptide can be orally administered or parenterally, topicallyadministered together with an adjuvant such as aluminum hydroxide, alumor muramyl dipeptide which is a synthetic adjuvant. It is possible toconduct the parenteral, topical administration, for example, throughsubcutaneous, intracutaneous, intramuscular or intravenousadministration, but since as to periodontal disease, immune response atthe limited site of the local gingiva is known, administration into thegingiva can also be adopted. As to the adjuvant, it is desirable to addaluminum hydroxide gel so that its final concentration can be 0.05 to0.2 mg/ml. It is suitable that the content of the peptide in the vaccineto be administered at a time is 0.004 to 2.5 mg, preferably 0.02 to 0.6mg. It is preferred to add 0.01% (w/v) of thimerosal as an antiseptic.

Further, apart from directly using the peptide as a vaccine, it is alsopossible to use, for prophylaxis or treatment of periodontal disease, anoral cavity composition containing a specific antibody found in theserum, cow's milk or eggs obtained after an ox, chicken or the like isimmunized with the peptide.

As for a method of immunizing a mammal with a peptide of the invention,as usually conducted, for example, a complex obtained by binding thepeptide to a carrier such as bovine serum albumin or tetanus toxoid canbe administered together with an adjuvant such as Freund's completeadjuvant or aluminum hydroxide to a living body.

As mammals to be immunized, there can be used goat, sheep, horse,cattle, etc. When antiserum or milk containing a specific antibodyobtained by immunizing a mammal with a complex of the peptide with thecarrier is used, several kinds of antisera or milks containing aspecific antibody can be mixed.

As to preparation of an antibody against the peptide through hen's eggs,for example, a specific antibody can be prepared by immunizing a chickenonce or twice with a complex obtained by binding the peptide to acarrier such as bovine serum albumin or tetanus toxoid, together with anadjuvant such as Freund's complete adjuvant or aluminum hydroxide,collecting, from one to two months later, eggs, separating yolk, addingphosphate-buffered physiological saline and chloroform to the yolk,stirring the mixture, and separating the water soluble part as the upperlayer which contains the specific antibody.

An antibody against the peptide, prepared by the invention, can becompounded into emulsions, dispersions, pastes, gels or aerosols.Specifically, the antibody can be compounded into substances such astoothpastes, chewing gums, mouthwashes or gargles.

Further, the peptides of the invention can be used for preparing aspecific antibody, and the resultant specific antibody can be utilizedfor diagnosis of periodontal disease, or for passive immunity as aprophylactic means against infection with Porphyomonas gingivalis.

Still further, the peptides of the invention can be used for producingpolyclonal antibodies or monoclonal antibodies. For obtaining polyclonalantibodies, either of large and small animals usually used can be used.After a host animal is immunized with such a peptide, an antibody can berecovered by a known method. On the other hand, a monoclonal antibodycan be obtained by immunizing a mouse with the peptide, and fusing aB-lymphocyte thereof and a myeloma cell.

The invention is specifically described below by examples, but it shouldnot be construed that the invention claimed in the attached CLAIM islimited thereby.

EXAMPLE 1 Synthesis of Partial Peptides Using Multi Pin PeptideSynthesis Kit

Peptides composed of 10 amino acid residues were synthesized, based onthe estimated amino acid sequence of the fimbriae of Porphyomonasgingivalis strain 381 determined by D. P. Dickinson et al. (J.Bacteriol. 170, No. 4, 1658-1665, 1988), at intervals of 5 amino acidresidues from the N-terminus side, using MULTI-PIN PEPTIDE SYNTHESIS KIT(Non-Cleavable Type: Code No. 593-28201) made by CHIRON MIMOTOPES PTYLTD (Australia).

First, the tip of the pin was immersed in dimethylformamide (DMF) for 2minutes, and washed by shaking three times in methanol (MeOH) for 10minutes. The tip was air dried for 30 minutes, and then immersed in 20%piperidine/DMF for 30 minutes to conduct deprotection. Then, the tip wasimmersed in DMF for 10 minutes, and washed by shaking 4 times inmethanol (MeOH) for 2 minutes. The tip was air dried for 30 minutes,immersed in DMF for 5 minutes, and then immersed in each Fmoc-amino acidsolution (60 mM)/DMF+120 mM 1-hydroxybenzotriazole monohydrate (HoBt),and reaction was conducted at room temperature for 16 hours to couplethe tip and the desired amino acid. From the next day, the couplingoperations were repeated in the same manner as above, using the nextamino acid.

When the coupling up to the desired 10 amino acid residues wascompleted, the tip was immersed in DMF for 2 minutes to swell it, andthen washed by shaking it three times in MeOH for 10 minutes. After airdrying for 30 minutes, the tip was immersed in 20% piperidine/DMF for 30minutes to conduct deprotection. Then, the tip was immersed in DMF for10 minutes, and washed by shaking it 4 times in MeOH for 2 minutes.After air drying for 30 minutes, the tip was immersed in a mixed liquidof DMF:acetic anhydride:triethylamine=50:5:1 for 90 minutes to conductacetylation, and the tip was washed by shaking it in MeOH for 2 minutes.After air drying for 15 minutes, the tip was immersed in a mixed liquidof trifluoroacetic acid:ethanedithiol:thioanisole=95:2.5:2.5 for 60minutes to conduct deprotection. The tip was air dried for 10 minutes,subjected to ultrasonic cleaning for 15 minutes, and further air driedfor 10 minutes.

Test Example 1 Elucidation of the Epitope Using Serum of a Patient ofPeriodontal Disease

Elucidation of the epitope was conducted by an ELISA method, using thepeptides synthesized in Example 1 and serum of a patient withperiodontal disease.

First, the tip of the pin to which the peptide is bound was blocked byimmersing in 2% bovine serum albumin (BSA)/phosphate-bufferedphysiological saline+Tween 20 (PBST) at room temperature for one hour,and then washed by shaking it three times in PBST for 5 minutes. Then,serum of a normal subject or the patient, on which addition of thefimbriae of Porphyomonas gingivalis and absorption operation wereconducted or not conducted, were prepared as primary antibodies. Each ofthe serum was diluted 10⁵ times with 0.1% casein/PBST, and reacted withthe peptide bound to the tip at 4° C. for 16 hours. The tip was washedby shaking it three times in PBST for 5 minutes, and then alkalinephosphatase-labeled anti-human IgG antibody (diluted 10³ times)/0.1%casein/PBST as a secondary antibody was reacted therewith. The tip waswashed by shaking three times in PBST for 5 minutes, and subjected toreaction in p-nitrophenyl phosphate/diethanolamine buffer at 37° C. for3 hours, and absorbance (405 nm) was measured. Peptides having adifference in absorbance between before and after the absorptionoperations of 0.2 or more were selected. Each of these peptides wassubjected to ultrasonic cleaning in 0.1 M phosphate buffer (pH 7.2)+1%SDS+0.1% 2-mercaptoethanol (55-65° C.), washed twice in distilled water(60° C.) for 30 seconds, and further washed by shaking for 30 minutes.Then, the peptide was boiled in methanol for 15 seconds, air dried for15 minutes, and then provided for a next ELISA method.

As a result, peculiar epitopes reacting with the serum of the patientwere found out. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                         Particularpeptides reacting with the serum                                     of a patient with periodontal disease                                         Amino acid sequence          Abbreviation                                   ______________________________________                                        Glu Asn Ala Thr Lys Val Glu Asp Ile Lys:                                                                  Peptide 1                                            - Glu Val Lys Ala Leu Thr Thr Glu Leu Thr: Peptide 2                          - Ala Glu Asn Gln Glu Ala Ala Gly Leu Ile: Peptide 3                          - Ala Ala Gly Leu Ile Met Thr Ala Glu Pro: Peptide 4                          - Thr Gly Ser Leu Thr Thr Phe Asn Gly Ala: Peptide 5                          - Thr Phe Asn Gly Ala Tyr Thr Pro Ala Asn: Peptide 6                          - Gly Phe Tyr Val Leu Glu Asn Asp Tyr Ser: Peptide 7                          - Ala Asn Gly Gly Thr Ile His Pro Thr Ile: Peptide 8                          - Glu Gly Lys Thr Tyr Tyr Pro Val Leu Val: Peptide 9                       ______________________________________                                    

EXAMPLE 2 Synthesis of Peptides Using a Peptide Synthesizer

Each of the particular epitopes reacting with the serum of the patientof periodontal disease, found out in Test example 1, was synthesizedaccording to R. B. Merrifield (J. Am. Chem. Soc. 85, 2149-2154, 1963)using a model 9050 peptide synthesizer (Millipore corporation).Purification was conducted by reverse-phase HPLC using Capcell Pak C18SG 120 (1.5×1.5 cm: Shiseido) column and an acetonitrile concentrationgradient of 6 to 60%, and thereby, the peptide fraction was recovered asthe main peak. As to each peptide thus prepared and purified, itscomposition was ascertained by quantitative amino acid analysis, and itsamino acid sequence by automated Edman degradation using a model6400/6600 protein sequencer (Japan Millipore corporation).

Test Example 2 Assay of the Specific Antibody in Serum of a Patient withPeriodontal Disease Using the Peptides

One mg of each peptide synthesized in Example 2 was dissolved in 1 ml of0.1 M phosphate buffered saline (PBS, pH 6.0), 5 mg of sulfated SMCC(made by Pierce Co.) was added, and the mixture was incubated at 30° C.for 30 minutes to conduct maleimidization, and then subjected toSephadex G-10 column chromatography, and the first eluate fraction wascollected. 7 mg of bovine serum albumin (BSA: made by Wako Pure ChemicalIndustries, Ltd.) was dissolved in 1 ml of 0.1 M phosphate buffer (pH6.0), 10 mM EDTA, the solution was mixed with the above maleimidizedpeptide, and the mixture was subjected to reaction at 30° C. for 1 hour.The resultant peptide-modified BSA was subjected to Sephadex G-50 columnchromatography, and the first eluate fraction was collected as aplate-preparing antigen solution.

Then, six kind of peptides were synthesized after the process of Example2, and a plate-preparing antigen solution was similarly prepared as acontrol.

Each of the resultant antigen solutions was diluted with 0.1 M carbonatebuffer (pH 9.6), 150 mM NaCl to give 100 ml of a solution. 10 μl/well ofthe solution was put in a polystyrene-made microtiter plate (ImmunoplateII; maxisorp type; made by Nunc Co.) and left alone at 4° C. overnight,and washing was conducted three times each with 200 μl/well of PBS,using a microtiter automatic washer (hereafter abbreviated as washer;made by Bio-Rad Laboratories). 250 μl/well of PBS containing 0.1%gelatin (made by Nitta Gelatin Co., Ltd.) was added and the mixture wasallowed to stand at 4° C. overnight to block the plate. The blockingsolution was removed by absorption, and the plate was dried by a vacuumdryer, put in an aluminum bag, and after sealing the bag, kept at 4° C.for the following assay.

Sera of patients of periodontal disease (n=3) and normal subjects (n=3)were used as samples, and each serum was diluted 100 times with PBScontaining 10% bovine serum. 100 μl portions of these dilutions wereadded to the plates coated with various antigens, respectively, andincubation was conducted at 37° C. for 1 hour. Each plate was washedthree times with 200 μl of PBST using the washer, 100 μl of acommercially available peroxidase-labeled human IgG antibody (diluted10³ times)/0.1% Casein/PBST was added as a secondary antibody, andincubation was conducted again at 37° C. for 1 hour. The plate waswashed in the same manner as above, 100 μl of an o-phenylenediaminesolution was added, incubation was conducted at 37° C. for 30 minutes,100 μl of 0.1 N sulfuric acid was added to stop the reaction, and then,absorbance (405 nm) was measured by a microplate reader (made by Bio-RadLaboratories) using the reaction well of no addition of the specimen asa control. The results of assay using the various antigen plates areshown in Table 2. The measures values are average absorbances of 3specimens each of the patient group of periodontal disease and thenormal subject group.

On the peptides having the amino acid sequences shown in Table 1 amongthe used peptides, specific reactivity was observed in the patient groupof periodontal disease. Therefore, it was revealed that by using thesepeptides as antigens, it is possible to detect anti-Porphyomonasgingivalis fimbria antibodies contained in sera of patients withperiodontal disease, and these peptides are useful for screening ofcases with periodontal disease.

                                      TABLE 2                                     __________________________________________________________________________    Assay of antibody titers of the sera of the periodontal                         disease patient group and normal subject group using                          plates coated with various peptides                                                             Patient group                                                with peridontal Normal subject                                               Peptides applied disease group                                              __________________________________________________________________________    Peptide of the invention                                                        Peptide 1 1.016 0.145                                                         Peptide 2 0.566 0.205                                                         Peptide 3 0.623 0.214                                                         Peptide 4 0.941 0.197                                                         Peptide 5 0.760 0.165                                                         Peptide 6 0.675 0.141                                                         Peptide 7 1.224 0.226                                                         Peptide 8 0.935 0.182                                                         Peptide 9 0.546 0.186                                                         Comparative peptide                                                           Val Met Val Tyr Asn Gly Glu Gln Gln Glu 0.165 0.172 (SEQ ID NO: 11)                                      Arg Thr Leu Val Val Met Ala Asn Thr Gly                                      0.199 0.161 (SEQ ID NO: 12)                         Asn His Ile Glu Asn Asp Pro Leu Lys Ile 0.205 0.201 (SEQ ID NO: 13)                                      Asp Ala Asn Tyr Leu Thr Gly Ser Leu Thr                                      0.196 0.145 (SEQ ID NO: 14)                         Trp Leu Ser Arg Asn Tyr Val Ala Pro Ala 0.210 0.204 (SEQ ID NO: 15)                                      Leu Cys Val Tyr Gly Lys Leu Gln Lys Asn                                      0.182 0.213 (SEQ ID NO: 16)                       __________________________________________________________________________

Test Example 3 Inhibitory Test of Porphyomonas Gingivalis InfectionUsing the Peptides

Each peptide synthesized in Example 2 was administered together with FIA(Freund's incomplete adjuvant) or FCA (Freund's complete adjuvant), as awater-in-oil type emulsifier, into the sole of the right hind leg of aguinea pig in an amount of 500 μg/animal/time. Thereafter, on the 28thday, 200 μl of a suspension of 10¹¹ cells/ml of the bacterium wasintracutaneously inoculated into the right flank of the same guinea pigafter the hair at the region was shaved. 24 hours later, the longestlength and shortest length of the resultant red coloring were measured,and the product of them was calculated. From the product was calculatedthe inhibition rate. These results are shown in Table 3, and as seentherefrom, particularly, Peptide 1 and Peptide 7 exhibited stronginhibitory effect against infection with Porphyomonas gingivalis.

                  TABLE 3                                                         ______________________________________                                        Inhibitory effect against infection with Porphyromonas gingivalis              using peptides                                                                                   Degree of redness                                                             Longest length ×                                       shortest length*) Inhibition                                                 Test group (mm.sup.2) rate (%)                                              ______________________________________                                        FIA.sup.a)          1947        0                                               FCA.sup.b) 1784 8.3                                                           Porphyromonas gingivalis fimbriae + FIA 1571 19.3                             Porphyromonas gingivalis fimbriae +  1486 23.7                                FCA                                                                           Peptide 1 + FCA  660 66.1                                                     Peptide 2 + FCA 1179 39.5                                                     Peptide 3 + FCA 1155 40.7                                                     Peptide 4 + FCA  851 56.3                                                     Peptide 5 + FCA 1063 45.4                                                     Peptide 6 + FCA 1112 42.9                                                     Peptide 7 + FCA  240 87.7                                                     Peptide 8 + FCA 1022 47.5                                                     Peptide 9 + FCA 1265 35.0                                                   ______________________________________                                         .sup.a) FIA: Freund's incomplete adjuvant                                     .sup.b) FCA: Freund's complete adjuvant                                       ##STR1##                                                                      A: Longest length × shortest length in the FIA inoculation group        B: Longest length × shortest length in the peptide inoculation grou     in the invention                                                         

Test Example 4 Test on Immunization of Mice with the Peptides

Each peptide synthesized in Example 2 was dissolved in PBS so as to makethe concentration 200 μg/ml, the solution was mixed with the same volumeof FCA, and the mixture after emulsification was subcutaneously injectedinto mice at the back (3 animals per group). 4 weeks later, the antibodytiter in the serum or saliva against the peptide was assayed by an ELISAmethod.

The fimbrial protein of Porphyomonas gingivalis was adsorbed on eachwell of a 96-well microtiter plate for ELISA, and a dilution of theserum with PBS (1/10⁵), a dilution of the saliva with PBS (1/500), or asolution obtained by adding each peptide or the fimbrial protein ofPorphyomonas gingivalis is to the dilution of the serum or saliva tocause absorption was put in the well. Reaction was conducted at 37° C.for 1 hour, and then assay was conducted by a usual ELISA method.

The results are shown in Table 4. As apparent therefrom, when the serumor saliva was subjected to absorption operations with each peptide,decrease of absorbance was observed. From the above results, it wasascertained that an antibody against each peptide or the fimbrialprotein was produced in the serum and saliva of the mice immunized witheach peptide.

                                      TABLE 4                                     __________________________________________________________________________    Immunization test with peptides                                                                Antibody titer before and after                                absorption operation.sup.c)                                                                  Serum      Saliva                                               Body (1/10.sup.5) (1/500)                                                             weight                                                                              Before                                                                             After Before                                                                             After                                          Test group (Average g) absorption absorption absorption absorption          __________________________________________________________________________    Fimbrial protein + FCA.sup.d)                                                            25    1.10 0.14  1.22 0.19                                           Control 27 0.08 0.07 0.18 0.16                                                Peptide 1 + FCA 25 0.62 0.19 0.61 0.11                                        Peptide 2 + FCA 26 0.34 0.15 0.36 0.17                                        Peptide 3 + FCA 26 0.38 0.16 0.37 0.09                                        Peptide 4 + FCA 27 0.52 0.13 0.49 0.12                                        Peptide 5 + FCA 28 0.45 0.11 0.42 0.15                                        Peptide 6 + FCA 26 0.41 0.12 0.39 0.11                                        Peptide 7 + FCA 23 0.67 0.14 0.69 0.15                                        Peptide 8 + FCA 25 0.49 0.11 0.45 0.14                                        Peptide 9 + FCA 26 0.32 0.13 0.34 0.13                                      __________________________________________________________________________     .sup.c) Antibody titer: A.sub.405 value by the ELISA method at each           dilution rate                                                                 .sup.d) FCA: Freund's complete adjuvant                                  

Test Example 5 Investigation of Various Immunobiological ActivitiesUsing the Peptides

On 9 peptides shown in Test example 1, Test example 2, Test example 3and Test example 4, mitogen activity, polyclonal B-cell activation,abilities to induce production of inflammatory cytokines such asTNF-alpha and IL-6 and hemagglutinating activity were assayed asbiological activities. Assay methods of various biological activitiesare shown below.

Mitogenic Activity

Spleen cells of BALB/c mice were cultured together with each of the ninepeptides obtained in Example 2 for 48 hours, and ³ H-thymidine was added6 hours before the final culture. After completion of the culture,uptake of ³ H-thymidine by the cells was measured using a scintillationcounter to examine the mitogen activity of each peptide.

Polyclonal B-Cell Activation

Spleen cells of BALB/c mice were cultured together with each of the ninepeptides obtained in Example 2 for 72 hours, and the number ofantibody-producing cells in the spleen cells was measured by an ELISPOTmethod to examine the polyclonal B-cell activation of each peptide.

Inductive Ability of TNF-alpha Production

Macrophages in human peripheral blood were cultured together with eachof the nine peptides obtained in Example 2 for 24 hours. Aftercompletion of the culture, the culture supernatant was added to L929cells for further 24 hours, and then the number of L929 dead cells wasmeasured to examine inductive ability the TNF-alpha production by eachpeptide.

Inductive Ability of IL-6 Production

Macrophages in human peripheral blood were cultured together with eachof the nine peptides obtained in Example 2 for 24 hours. Then, theamount of IL-6 in the culture supernatant was assayed by an ELISA methodto examine the inductive ability of IL-6 production by each peptide.

Hemagglutinating Activity

0.1 ml portions of each of solutions of a peptide 2-fold seriallydiluted in PBS were put in each well of a 96-well round-bottomedmicrotitration plate, and 0.1 ml portions of a suspension of 2% rabbiterythrocytes were added, respectively. After culture at 37° C. for 2hours, the hemagglutinating pattern of each well was estimated.

The results of the above tests are shown in Table 5, and as apparenttherefrom, Peptide 1 and Peptide 7 do not exhibited any of theseactivities.

From these results, Peptide 1 and Peptide 7 not having any of thevarious immunobiological activities were decided to be used as a vaccinefor prophylaxis of periodontal disease.

                  TABLE 5                                                         ______________________________________                                        Various immunobiological activities of peptides                                               Polyclonal             Hema-                                    Mitogenic B-cell TNF-alpha IL-6 gglutinating                                  activity activation production production activity                          ______________________________________                                        Peptide 1                                                                            -        -        -      -      -                                        Peptide 2 + + + + ±                                                        Peptide 3 - - + + ±                                                        Peptide 4 - - + + -                                                           Peptide 5 N.D.e) N.D. N.D. N.D. +                                             Peptide 6 N.D. N.D. N.D. N.D. +                                               Peptide 7 - - - - -                                                           Peptide 8 N.D. N.D. N.D. N.D. ±                                            Peptide 9 N.D. N.D. N.D. N.D. ±                                          ______________________________________                                         e) N.D.: Not tested                                                      

EXAMPLE 3 Preparation of a Vaccine

Peptide 1 or Peptide 7 among the peptides prepared in Example 2 wasdissolved in 0.75 M phosphate-buffered physiological saline so that theconcentration of the peptide could be 1.0 mg/ml, aluminum hydroxide gelwas added as an adjuvant so that the concentration could be 0.2 mg/ml,and 0.01% (w/v) of thimerosal was added to give a vaccine.

Test Example 6 Inhibitory Test of Porphyomonas gingivalis Infection

The effects of vaccine on Peptide 1 and Peptide 7 selected from theresults of Test example 1, Test example 2, Test example 3, Test example4 and Test example 5 were examined according to the following methodusing degree of resorption of alveolar bone and antibody titer asmeasures, using the vaccine described in Example 3.

3-week-old Golden hamsters (one group consisting of 6 animals, 5 groupsin total, 1 Porphyomonas gingivalis strain 381 fimbria immunityinduction group, 2 Peptide 1 immunity induction group, 3 Peptide 7immunity induction group, 4 Nonimmunity induction group, 5 Nonimmunitynoninduction group) were used.

Each of the vaccines of the invention was subcutaneously administered atthe oral cavity in amounts of 0.2 ml portions on the third week and thefourth week after the birth, and 0.4 ml on the fifth week.

Induction of periodontal disease was conducted by culturing Porphyomonasgingivalis strain 381 in GAM broth at 37° C. for 20 hours, collectingthe cells by centrifugation, suspending the cells in phisiologicalsaline so that the cell concentration could be 10⁹ cells/ml,administering 0.2 ml portions of the suspension into the oral cavity ofthe Golden hamsters for 7 days starting from the sixth week after thebirth, and thereafter, administering the suspension once a week.

After the start of the cell administration, the animals were allowed tofreely ingest Diet 2000 (made by Funabashi Nojo) as a dentalcaries-inducing feed and water.

After the Golden hamsters were fed for 80 days, 1 ml/kg portions of 0.5%pilocarpine hydrochloride were intraperitoneally administered, andsaliva was taken from them. Thereafter, blood was taken from theabdominal aorta of each animal, and serum was obtained therefrom.Further, a bone preparation from each hamster was produced by amputatingthe head, heat treating the head using an autoclave, removing the musclepart, and washing with water and drying the remaining matter.

The saliva and serum were assayed for antibody titer against thepeptide.

The bone preparation was assayed for degree of resorption of thealveolar bone of a molar according to the method^(f)) of Tsukiyama etal. (Koku Eisei Kaishi (Jornal of Hygiene of Oral Cavity) 28, No. 3,149, 1978).

f) The bone preparation was stained with 20% silver nitrate solution for5 minutes, and after water washing and drying, the distance from theenamel-cement junction to the alveolar bone edge was measured by astereoscopic microscope (magnification 15-fold).

In the inhibitory test of Porphyomonas gingivalis infection, as seen inTable 6, strong resorption of the alveolar bone was observed in thenonimmunity induction group, but in the immunity induction groups, thedegree of resorption of the alveolar bone was inhibited in almost thesame degree as in the nonimmunity noninduction group.

As to antibody titers in the sera and saliva of the test groups againstthe peptides, the antibody titers were significantly incresed in thepeptide administration groups, but the antibody titer in the nonimmunityinduction group was almost equal to that in the nonimmunity noninductiongroup.

These results are shown in Table 6, and as seen therefrom, in Peptide 1and Peptide 7 were observed inhibition of absoption of the alveolarbone, and increse of antibody titer in the sara and saliva.

                                      TABLE 6                                     __________________________________________________________________________    Inhibitory test of Porphyromonas gingivalis infection in Golden hamster                       Body weight (at the                                                                            time of production Absorbance of                                             Antibody titer.sup.g)                                         of bone preparation                                                                    alveolar bone                                                                        Serum                                                                             Saliva                                      Test group (average g) (average) (1/10.sup.5) (1/500)                       __________________________________________________________________________    Fimbria immunity induction group                                                              128      52     1.08                                                                              1.29                                        Peptide 1 immunity induction group 124 48 0.65 0.62                           Peptide 7 immunity induction group 123 41 0.71 0.79                           Nonimmunity induction group 116 97 0.10 0.21                                  Nonimmunity noninduction group 135 34 0.06 0.16                             __________________________________________________________________________     .sup.g) Antibody titer: A.sub.405 value by the ELISA method at each           dilution rate                                                            

Test Example 7 Safety of the Vaccine for Prophylaxis of PeriodontalDisease

The vaccines obtained in Example 3 were tested by the staining test,aseptic test and acute toxicity test (mice) according to the A TestMethods in Biological Preparation Standards notified by the Ministry ofWelfare, but abnormality to be specially mentioned was not recognized inany of the tests.

EXAMPLE 4 Obtention of Murine Antisera Against the Peptides

BALB/c mice were immunized with 300 μg portions of each of the peptideswhose effects were ascertained in Test example 3, together with FCA,respectively, to give antisera against the respective peptides. In thesame manner as above, antisera against the whole cell of Porphyomonasgingivalis (100 μg/time) and the fimbria (100 μg/time) were obtained,respectively.

The respective antisera against the peptides, whole cell and fimbriawere purified through ammonium salfate fractionation and ion exchangechromatography, and antibody solutions each having a protein content of1 mg/ml were prepared.

Test Example 8 Cell Agglutination Test Using Antibodies Against thePeptides

Porphyomonas gingivalis was inoculated in a medium comprising GAM broth,Brain-Heart Infusion broth or the like having added thereto hemin andmenadione and cultured under an anaerobic condition at 37° C. for 20hours, the cells were collected and suspended in phosphate-bufferedphysiological saline, and the degree of resorption of the suspension(550 nm) was adjusted so as to be 0.5. 100 μl portions of the cellsuspension and 100 μl portions of dilutions of each antibody solutionobtained in Example 4 were adequately mixed, respectively, and themixtures were put in a 96-well round-bottomed microtitration plate andsubjected to reaction at 4° C. for 16 hours, respectively, to conductcell agglutination test. The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Cell agglutination test                                                                    Dilution rate of antibody                                                         10.sup.-1                                                                            10.sup.-2                                                                           10.sup.-3                                                                          10.sup.-4                                                                          10.sup.-5                                                                           10.sup.-6                       ______________________________________                                        Anti-entire cell                                                                           +++    +++     +++  ++   +     -                                   antiserum                                                                     Anti-fimbria serum +++ +++ ++ + - -                                           Anti-Peptide 1 serum +++ +++ ++ - - -                                         Anti-Peptide 7 serum +++ +++ ++ - - -                                       ______________________________________                                    

EXAMPLE 5

    ______________________________________                                        Preparation of toothpaste                                                     ______________________________________                                        Calcium secondary phosphate dihydride                                                                   45%                                                   Glycerol 15%                                                                  Sorbitol 10%                                                                  Carboxymethyl cellulose 5%                                                    Sodium lauryl sulfate 3%                                                      Saccharin 0.1%                                                                Water 21.9%                                                                    100%                                                                       ______________________________________                                    

0.01% of the antibody solution against Peptide 1 obtained in Example 4is incorporated into the above components.

EXAMPLE 6

    ______________________________________                                        Preparation of mouthwash                                                      ______________________________________                                        Ethanol                 22%                                                     Saccharin 0.1%                                                                Lauryldiethanolamine 0.3%                                                     Flavor 1%                                                                     Water 76.6%                                                                    100%                                                                       ______________________________________                                    

0.01% of the antibody solution against Peptide 7 obtained in Example 4is incorporated into the above components.

Test Example 9 Passive Immunity Test Using Antibodies Against thePeptides

Effect of passive immunity on Golden hamsters was examined by rinsingthe mouth and brushing the teeth using the antibody solutions againstthe peptides obtained in Example 4.

Effect of passive immunity by the invention was examined according tothe following method using degree of resorption of the alveolar bone andantibody titer as measures. 3-week-old Golden hamsters (one group 6animals, 4 groups, 1 Anti-Peptide 1 antibody administration inductiongroup, 2 Anti-Peptide 7 antibody administration induction group, 3Nonpassive immunity induction group, 4 Nonpassive immunity noninductiongroup) were used.

Induction of periodontal infection was conducted by culturingPorphyomonas gingivalis strain 381 in GAM broth at 37° C. for 20 hours,collecting the cells by centrifugation, suspending the cells inphisiological saline so that the cell concentration could be 10⁹cells/ml, administering 0.2 ml portions of the suspension into the oralcavity of the Golden hamsters wherein the first molar of the lower jawwas ligated with cotton thread.

After the start of the cell administration, the animals were allowed tofreely ingest Diet 2000 (made by Funabashi Nojo) as a dentalcaries-inducing feed and water.

From the next day of the cell administration, rinse of the mouth and thebrushing were conducted every day using the oral cavity compositioncontaining the antibody against the peptide, obtained in Example 5.

A bone preparation from each hamster was produced by amputating thehead, heat treating the head using an autoclave, removing the musclepart, and washing with water and drying the remaining matter.

As to infection protection effect by passive immunity, as shown in Table8, strong degree of resorption of the alveolar bone was observed in thenonpassive immunity induction group, but in the anti-Peptide 1 antibodyadministration induction group and the anti-Peptide 7 antibodyadministration induction group, degree of resorption of the alveolarbone was inhibited to almost the same degree as in the nonpassiveimmunity noninduction group.

                                      TABLE 8                                     __________________________________________________________________________    Passive immunity test using Golden hamsters                                                     Body weight at the time                                       of production of bone Absorbance of                                           preparation alveolar bone                                                     (average g) (average)                                                       Test group        Mouth rince                                                                         Brushing                                                                           Mouth rince                                                                         Brush                                      __________________________________________________________________________    Anti-Peptide 1 antibody adminstration                                                           120   122  60    51                                           induction group                                                               Anti-Peptide 7 antibody administration 120 123 56 46                          induction group                                                               Nonpassive immunity inductioin group 118 116 98 97                            Nonpassive immunity noninduction group 122 124 41 39                        __________________________________________________________________________

Industrial Applicability

According to the invention, by synthesizing peptides of the 41 kDsubunit protein constituting the fimbriae of Porphyomonas gingivalis,and utilizing the peptides as a antigen, it is possible to accuratelyassay specific antibodies in the sera, saliva and gingival crevice fluidof patients with periodontal disease, and further their classes orsubclasses.

Further, when a peptide of the 41 kD subunit protein constituting thefimbriae of Porphyomonas gingivalis is synthesized, and the peptide isadministered together with a suitable immunopotentiating agent into thegingiva, a specific antibody is effectively increased and infection withPorphyomonas gingivalis is inhibited. Further, it is also possible touse an oral cavity composition containing an antibodies contained inserum, milk or egg obtained by immunizing an ox, chicken or the likewith such a peptide, for prophylaxis or treatment of periodontaldisease.

Thus, the present invention can be utilized in the manufacturingindustry of diagnostic reagents, the manufacturing industry of vaccines,etc.

    __________________________________________________________________________    -                   - #             SEQUENCE LISTING                             - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  16                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Glu Asn Ala Thr Lys Val Glu Asp Ile Lys                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Glu Val Lys Ala Leu Thr Thr Glu Leu Thr                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Ala Glu Asn Gln Glu Ala Ala Gly Leu Ile                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ala Ala Gly Leu Ile Met Thr Ala Glu Pro                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Thr Gly Ser Leu Thr Thr Phe Asn Gly Ala                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Thr Phe Asn Gly Ala Tyr Thr Pro Ala Asn                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Gly Phe Tyr Val Leu Glu Asn Asp Tyr Ser                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Ala Asn Gly Gly Thr Ile His Pro Thr Ile                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Glu Gly Lys Thr Tyr Tyr Pro Val Leu Val                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  5 amino - # acids                                                (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Asn Ala Thr Lys                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Val Met Val Tyr Asn Gly Glu Gln Gln Glu                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Arg Thr Leu Val Val Met Ala Asn Thr Gly                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Asn His Ile Glu Asn Asp Pro Leu Lys Ile                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Asp Ala Asn Tyr Leu Thr Gly Ser Leu Thr                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Trp Leu Ser Arg Asn Tyr Val Ala Pro Ala                                   1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  10 amin - #o acids                                               (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Leu Cys Val Tyr Gly Lys Leu Gln Lys Asn                                   1              5   - #                 10                                  __________________________________________________________________________

What is claimed is:
 1. An isolated peptide consisting of 5 or 10 successive amino acid residues in the amino acid sequence of SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8 or 9, or a salt of the isolated peptide, said isolated peptide having specific reactivity with serum of a periodontal disease patient.
 2. A composition comprising one or more isolated peptide(s) or salt(s) thereof, wherein each peptide consists of 5 or 10 successive amino acid residues in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9, said isolated peptide(s) or salt(s) thereof having specific reactivity with serum of a periodontal disease patient.
 3. The composition according to claim 2, which is for the diagnosis of periodontal disease.
 4. A vaccine comprising one or more isolated peptide(s) or salt(s) thereof, and one or more pharmaceutically acceptable carriers, said isolated peptide consisting of 5 or 10 successive amino acid residues in the amino acid sequence of SEQ ID No. 1 or 7 and having specific reactivity with serum of a periodontal disease patient.
 5. The vaccine according to claim 4, which is for the prophylaxis of periodontal disease.
 6. The vaccine according to claim 5, which is for administration into gingiva.
 7. An oral cavity composition for prophylaxis or treatment of periodontal disease comprising an antibody obtained by immunizing an animal with one or more isolated peptide(s) or salt(s) thereof, each of said isolated peptide consisting of 5 or 10 successive amino acid residues in the amino acid sequence of SEQ ID No. 1 or 7 and having specific reactivity with serum of a periodontal disease patient.
 8. A method for protecting a mammal from periodontal disease or for treating a mammal suffering from periodontal disease, which comprises administering the vaccine according to claim 4 to the mammal.
 9. The isolated peptide or salt thereof according to claim 1, wherein the peptide consists of the amino acid sequence as set forth in SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8 or
 9. 10. The vaccine according to claim 4, wherein at least one of the isolated peptide consists of the amino acid sequence as set forth in SEQ ID No. 1 or
 7. 11. The oral cavity composition according to claim 7, wherein at least one of the isolated peptide consists of the amino acid sequence as set forth in SEQ ID No. 1 or
 7. 12. The method according to claim 8, wherein at least one of the isolated peptide consists of the amino acid sequence as set forth in SEQ ID No. 1 or
 7. 13. An isolated peptide consisting of the amino acid sequence as set forth in SEQ ID No. 10 or a salt of the isolated peptide, said isolated peptide having a specific reactivity with serum of a periodontal disease patient. 